Method of inducing gene expression in plant and the plant treated thereby

ABSTRACT

The present invention is to provide a method which comprises providing a plant with characters of a repressor and operator both constituting a gene expression inducing system with an actinomycete autogenous regulatory factor as an inducer by gene transfer and administering the actinomycete autogenous regulatory factor to the transformed plant to thereby induce the expression of a gene placed under the control of the operator at a site of administration of the actinomycete autogenous regulatory factor. This method makes it possible to cause expression of a desired gene at a desired time and site, thus enabling even the production, in a plant, of a metabolite otherwise disadvantageous to the growth of the plant. It is also useful in preventing transformant plants from spreading through the environment by controlling the fertility thereof.

RELATED APPLICATIONS

This application is a nationalization of PCT application PCT/JP01/05096filed Jun. 15, 2001. This application claims priority from the PCTapplication and Japan Application Serial No. 2000-180466 filed Jun. 15,2000.

TECHNICAL FIELD

The present invention relates to a technique of providing a plant with agene expression inducing system through production of a transgenic plantutilizing the gene recombination technology.

BACKGROUND ART

To provide a plant with a novel character by transferring a gene intothe plant is called transformation. When the gene transferred isexpressed in plant cells, the character provided manifests itself. Oncethe gene has been integrated in an intracellular chromosome, thecharacter provided will be maintained stably. Such character to be newlyprovided by gene transfer includes, for example, resistance to diseasesand agricultural chemicals and changes in metabolism. Genes for use insuch transformation can freely be constructed using the current generecombination technology. Several methods have been developed fortransferring the genes constructed in such a manner into plants. Forefficiently integrating a gene into a plant cell nuclear chromosome,there is available the Agrobacterium infection method which utilizes, asa vehicle (vector) for the gene, Agtrobacterium, which is aplant-infective bacterium.

The expression of a gene involves a step, called transcription, in whichmRNA is transcribed upon a template, namely DNA which is the very genecontaining a genetic information, and a step, called translation, inwhich a protein is synthesized based on the genetic information from thetranscript mRNA. It is known that a gene comprises regions involved intranscriptional regulation or control in addition to the region encodingthe protein information. The most basic transcriptional regulatoryregion is a 5′ upstream region relative to the coding region and iscalled a promoter. The promoter differs in structure between eukaryotes,such as plants, and prokaryotes, such as bacteria. Plant promoters havea nucleotide sequence called TATA box, which is essential for initiatinggene transcription, and other various regulatory sequences. Forinitiating transcription, RNA polymerase, which is an enzyme catalyzingthe transcription in plant cells, binds to the TATA box. Variousintracellular proteins called transcription factors specifically bind tothe various regulatory sequences serving as targets for those factors.These transcription factors promote or inhibit the transcriptionalactivity of RNA polymerase and thereby control the gene expression.Thus, gene expression is under the control of such regulatory sequences.These regulatory sequences and transcription factors are also involvedin induction of gene expression via the step of transcription.

To control the induction of expression of a gene transferred into aplant for transformation with respect to time and site makes itpossible, with great advantage, to produce, in plants, such metabolitesas otherwise will be disadvantageous to plant growth. For such purposes,the utilization of a gene expression inducing system of other organismshas often been attempted. This is because the use of a gene expressioninducing system intrinsic in a plant as it is may possibly exert anunexpected influence on the metabolic system of the plant. However, itis not self-evident whether the gene expression inducing system of otherorganisms can be successfully given to the plant.

The regulatory system comprising an inducer, repressor and operator asfound in the bacterial operon regulatory system is one of the principalgene expression inducing systems. The inducer is a low-molecular-weightcompound inducing gene expression. The repressor is a receptor proteinfor the inducer. The operator is a regulatory sequence serving as atarget for the repressor. The inducer-repressor binding andrepressor-operator binding are very specific and show high levels ofaffinity, whereas the inducer-bound repressor cannot bind to theoperator. A gene containing the operator in its promoter, namely a geneunder the control of the operator, is inhibited (OFF) from beingexpressed when the inducer concentration is low because the repressor isbound to the operator but, as the inducer concentration increases, therepressor is released and gene expression is induced (ON).

Attempts have been reported to utilize the bacterialinducer/repressor/operator system as means for inducing gene expressionin plants. For providing a plant with the characters of a repressor andoperator, two genes, namely a repressor gene and a gene under thecontrol of an operator, are transferred into the plant. For attainingexpression of both genes in plant cells, it is desirable that thepromoter therefor be a plant promoter. The operator is located in andnear the plant promoter. By choosing the promoter, it is possible tofunctionally combine various characteristics of the promoter, such asgene expression intensity and tissue specificity, with the geneexpression inductivity. By administering an inducer to the planttransformed in this manner, the expression of the gene placed under thecontrol of the operator is induced at the site of administration of theinducer. As examples of the success in providing plants with suchinducer/repressor/operator regulatory systems, there are reports on thesystems in which tetracycline and IPTG are used as inducers [JapaneseKokai Publication Hei-06-339384 and Gatz et al., Trends in Plant Science(1998), 3, 352–358]. However, the inducer substances used in theexamples so far reported have problems from feasibility points of view,for example in the aspects of environmental safety and/or cost of use.

SUMMARY OF INVENTION

In view of the above state of the art, it is an object of the presentinvention to provide a method of inducing gene expression in a plant tothereby control the time and site of expression induction of a genetransferred into the plant for transformation.

The present invention is a method of inducing gene expression in a plant

which comprises providing the plant with characters of a repressor andoperator both constituting a gene expression inducing system with anactinomycete autogenous regulatory factor as an inducer by gene transferand

administering the actinomycete autogenous regulatory factor to thetransformed plant to thereby induce the expression of a gene placedunder the control of the operator at a site of administration of theactinomycete autogenous regulatory factor.

In the following, the present invention is described in detail.

DETAILED DISCLOSURE OF THE INVENTION

Actinomycetes occur in soils in the highest density next to eubacteriaand produce a number of physiologically active substances, such asantibiotics. As the actinomycetes, there may be mentioned, for example,the genera Streptomyces, Micromonospora, Actinomadura,Streptosporangium, Actinoplanes, Nocardia and Saccharopolyspora. Theproduction of physiologically active substances in and the morphologicaldifferentiation of actinomycetes are controlled by endogenous microbialhormone-like substances, namely autogenous regulatory factors.

So far, three actinomycete autogenous regulatory factors are known,namely A factor in Streptomyces griseus, virginiae butanolide (VB) inStreptomyces virginiae and Inducing Material-2 in strain FRI-5 ofStreptomyces lavendulae [Nihira, Hakko Kogaku Kaishi (1991), volume 69,89–105].

The A factor induces the production of the antibiotic streptomycin andthe streptomycin resistance in the producer and also induces theformation of conidiospores and aerial hyphae. VB induces the production,in the producer, of two species of the antibiotic virginiamycin, namelyvirginiamycin M and virginiamycin S, simultaneously. The InducingMaterial-2 induces the conversion in antibiotic production in theproducer (conversion from D-cycloserine to a nucleoside type antibiotic)and also induces the production of a blue pigment in a conditioninsufficient in carbon source and nitrogen source.

Like hormones, pheromones and the like as seen in other organismspecies, the actinomycete autogenous regulatory factors show theiractivity at very low concentrations, namely several nM to several scorenM, in culture.

About 60% of the actinomycetes belonging to the genus Streptomyces aresupposed to produce autogenous regulatory factors and there is thepossibility that a number of unknown autogenous regulatory factors stillexist.

All the known actinomycete autogenous regulatory factors has, in common,the 2-(1′-oxo or hydroxyalkyl)-3-hydoxymethyl-butyrolactone skeleton.Therefore, the known actinomycete autogenous regulatory factors are alsocalled butyrolactone autogenous regulatory factors. In all of them, thetwo substituents on the lactone ring are trans in the stereostructure toeach other and their absolute configurations are 2R and 3R. The factorsdiffer in three respects, namely the alkyl side chain at position 2, theposition 6, which is carbonyl or hydroxyl, and the orientation of thehydroxyl group, which is α (Inducing Material-2 type) or β (VB type).

Five VB species (A, B, C, D and E) having different alkyl side chains atposition 2 are known to exist. Artificially synthesized derivatives alsoshow the activity. The structure of the side chain at position 2influences the intensity of the activity.

The actinomycete autogenous regulatory factors are relatively simple instructure and therefore their chemical synthesis is easy. It is alsopossible to axenically cultivate the producer of each factor in largeamounts and separate and purify each factor from the culture thereof.

The occurrence of respective receptor proteins for A factor, VB andInducing Material-2 in respective producers has been established andthey have been named ArpA [Onaka et al., J. Bacteriol. (1995), 177,6083–6092], BarA [Okamoto et al., J. Biol. Chem. (1995), 270,12319–12326] and FarA [Waki et al., J. Bacteriol. (1997), 179,5131–5137], respectively. They are composed of 276, 232 and 221 aminoacids, respectively. In each of the receptor proteins, there is found,at the N terminus thereof, a helix-turn-helix motif indicative of theDNA binding ability.

The amino acid sequence of BarA, for instance, and the nucleotidesequence coding for the same are represented by SEQ ID NO:1 and SEQ IDNO:2, respectively.

The specific binding affinity between an actinomycete autogenousregulatory factor and its receptor protein is very high and thedissociation constant (Kd value) thereof is, for example, 0.7 nM for Afactor/ArpA, and 1.1 nM for VB-C₇/BarA [Nihira, Hakko Kogaku Kaishi(1991), vol. 69, 89–105].

For example, the occurrence has been made clear of genes named barB andbarX seemingly under the control of a gene expression inducing systemcommon to the barA gene coding for the receptor protein BarA for VB atsites 3′ downstream and 5′ upstream thereof. Although functions of theproteins encoded by these genes are not clear yet, they are supposedlyinvolved in virginiamycin biosynthesis in or virginiamycin resistance ofthe producer or in the regulatory system therefor.

As a result of an in vivo experiment [Kinoshita et al., J. Bacteriol.(1997), 179, 6986–6993], it was shown that BarA is a repressor bindingto the barA and barB gene promoters and shut OFF the transcription ofthese genes and that VB is an inducer causing BarA to depart from thepromoters to thereby turn ON the transcription of these genes. Further,as a result of an in vitro experiment [Kinoshita et al., J. Bacteriol.(1999), 181, 5075–5080], a target sequence (operator) to which BarAspecifically binds was identified on each of the barA and barB genes andnamed BARE. It includes BARE-3 (26 bp) on the barA gene promoter, andBARE-1 (29 bp) and BARE-2 (28 bp) on the barB gene promoter. Thenucleotide sequence of BARE-3, for instance, is shown under SEQ ID NO:3.

In this way, it was revealed that the actinomycete autogenous regulatoryfactor is involved in the gene expression inducing system in theproducer. This gene expression inducing system comprises an inducer,repressor and operator. The actinomycete autogenous regulatory factor,the receptor protein for the actinomycete autogenous regulatory factorand the target sequence for the receptor protein function as theinducer, repressor and operator, respectively.

In accordance with the present invention, a plant is provided withcharacters of a repressor and operator both constituting a geneexpression inducing system with an actinomycete autogenous regulatoryfactor as an inducer by gene transfer. The plant to be used in thepractice of the present invention includes tobacco, corn, soy, rape,potato, cotton and the like.

To provide a plant with a character of a repressor, so referred toherein, means transformation of the plant by transfer of a repressorgene into the same. To provide a plant with a character of an operatormeans transformation of the plant by transfer of a gene placed under thecontrol of an operator into the same.

In other words, in accordance with the present invention, two genes, agene for a receptor protein for an actinomycete autogenous regulatoryfactor and a gene placed under the control of a target sequence for thereceptor protein, are transferred into a plant for transformationthereof.

For transferring a gene for a receptor protein (repressor) for anactinomycete autogenous regulatory factor into a plant fortransformation thereof, the coding region of the receptor protein geneis connected to a site 3′ downstream of a promoter functioning in theplant and this is incorporated into an appropriate plasmid vector. Thepromoter to be used here is preferably a plant promoter.

For example, the use of the Cauliflower mosaic virus (CaMV) 35Spromoter, which is known to exhibit a potent promoter activity in avariety of plant species is effective in causing potent constitutivegene expression in plants. Other plant promoters include, but are notlimited to, Agrobacterium-derived opine (nopaline, octopine, mannopine)synthase gene promoters. Ordinary plant promoters can also be used.

To be transferred into a plant by the Agrobacterium infection method,for instance, a desired gene is incorporated into a plasmid vectorcalled binary vector. The binary vector has replication systemsfunctioning in Escherichia coli and Agrobacterium, a selective markergene and, in addition, 25 bp nucleotide sequences called RB and LB whichare essential for gene integration into a plant cell nuclear chromosome.The gene inserted between RB and LB of the binary vector, whentransferred into a plant cell, is efficiently integrated into a nuclearchromosome.

For example, a binary vector for transferring the gene for the receptorprotein (repressor) BarA for the actinomycete autogenous regulatoryfactor VB into a plant for transformation thereof can be constructed byconverting the coding region of the β-glucuronidase (GUS) gene of pBI121[Jefferson et al., EMBO J. (1987), 6, 3901–3907], which is a binaryvector having a structure such that the coding region of the GUS gene isconnected to a site 3′ downstream of the CaMV 35S promoter, or the likebinary vector to the coding region of the barA gene. When, for example,a barA gene coding region fragment having recognition sites for therestriction enzymes BamHI and SacI at both respective ends is prepared,the GUS gene coding region of the binary vector pBI121 can be convertedto the barA gene coding region through the aid of the restriction enzymeBamHI and SacI recognition sites. A barA gene coding region fragmenthaving the restriction enzyme BamHI and SacI recognition sites at bothrespective ends can be obtained, for example, by carrying out the PCRusing chemically synthesized oligo-DNAs respectively having thenucleotide sequences shown under SEQ ID NO:8 and SEQ ID NO:9 as 5′- and3′-PCR primers and the plasmid pET-p26k [Okamoto et al., J. Biol. Chem.(1995), 270, 12319–12326] containing the barA gene shown under SEQ IDNO:1 as a template.

For transferring a gene placed under the control of a target sequence(operator) for the actinomycete autogenous regulatory factor receptorprotein into a plant for transformation thereof, the target sequence isdisposed in a promoter functioning in the plant, the coding region of adesired arbitrary gene is connected to thus-modified promoter at a site3′ downstream thereof and the resulting structure is incorporated intoan appropriate plasmid vector. The promoter to be used here ispreferably a plant promoter.

The target sequence (operator) is desirably disposed in the vicinity ofa site 3′ downstream or 5′ upstream of a TATA box of the promoter and itis efficient to dispose the target sequence repeatedly.

For example, a binary vector for transferring the GUS gene placed underthe control of the target sequence (operator) BARE for the VB receptorprotein BarA into a plant for transformation thereof can be constructedby disposing the BARE sequence in the CaMV 35S promoter of the binaryvector pBI121 or the like. The enzyme encoded by the GUS gene can beeasily detected through its activity and the GUS gene has no homologuein plants and, therefore, the gene is widely used as a reporter gene forexperimentally detecting gene expression activity in plant cells. When,for example, the promoter has appropriate restriction enzyme recognitionsites between which the target sequence (operator) is to be disposed,the disposition of the target sequence in the promoter can be attainedby synthesizing a double-stranded DNA fragment comprising the nucleotidesequence between the restriction enzyme recognition sites. Thedisposition is also possible by the technique of site-directedmutagenesis which utilizes a chemically synthesized oligo-DNA. Fordisposing BARE-3 shown under SEQ ID NO:3 in the vicinity of a site 3′downstream or in the vicinity of a site 5′ upstream of the TATA box ofthe CaMV 35S promoter, for instance, a double-stranded DNA fragmentcomprising, for instance, the nucleotide sequence shown under SEQ IDNO:4 or 5 may be synthesized, respectively. For disposing BARE-3 in thevicinity of a site 3′ downstream and in the vicinity of a site 5′upstream of the TATA box of the CaMV 35S promoter, for instance, adouble-stranded DNA fragment comprising the nucleotide sequence shownunder SEQ ID NO:6, for instance, may be synthesized. For repeatedlydisposing BARE-3 in the vicinity of a site 3′ downstream or in thevicinity of a site 5′ upstream of the TATA box of the CaMV 35S promoter,for instance, a double-stranded DNA fragment comprising the nucleotidesequence shown under SEQ ID NO:7, for instance, may be synthesized. Forexample, for synthesizing a double-stranded DNA fragment comprising thenucleotide sequence shown under SEQ ID NO:7 in which two BARE-3sequences are disposed in the vicinity of a site 3′ downstream of theTATA box of the CaMV 35S promoter and one BARE-3 sequence in thevicinity of a site 5′ upstream of the same box, chemically synthesizedoligo-DNAs comprising the nucleotide sequences shown under SEQ ID NO:10and SEQ ID NO:11 whose 3′ termini are complementary to each other over16 bp are mixed up in a test tube and the complementary termini areallowed to anneal. DNA polymerase is added to this, and thedouble-stranded DNA fragment synthesized is treated with the restrictionenzymes EcoRV and XbaI and then cloned in an appropriate plasmid vector.

In accordance with the present invention, the plasmid vector constructedin the above manner is transferred into a plant for transformationthereof.

In gene transfer into a plant using the Agrobacterium infection method,for instance, Agrobacterium tumefaciens or Agrobacterium rhizogenes isfirst transformed by transfer of the binary vector constructed in theabove manner. For this transformation, the electroporation method or thelike method is effective. The Agrobacterium species to be used on thatoccasion is required to have a function necessary for the integration ofthe region sandwiched between RB and LB of the binary vector in a plantcell nuclear chromosome. The transformant Agrobacterium can be easilyselected by utilizing the function of a selective marker gene in thebinary vector. A plant is infected with the transformant Agrobacteriumthus obtained in which the binary vector containing the desired gene istransferred. For this purpose, a plant tissue section is cultured withthe transformant Agrobacterium. Then, a callus is induced from thetissue section. On that occasion, an antibiotic for killingAgrobacterium, for example carbenicillin, is caused to coexist inaddition to the selective marker agent. Utilizable as the selectivemarker gene are genes giving resistance to antibiotics, for example,kanamycin, hygromycin, bleomycin and chloramphenicol. Thus-obtainedtransformant callus is placed on a regeneration medium to allow plantregeneration to give a transformant plant. A transformant plant line canalso be obtained from seeds of the transformant plant.

Cultured plant cell transformants can also be obtained in the samemanner. In this case, however, it is not necessary to take the step ofcallus formation, plant body regeneration or seed formation or the likestep.

In addition to the Agrobacterium infection method, other methods of genetransfer into plants are also available, such as the electroporationmethod for transferring a gene into protoplasts, the particlebombardment method which uses a gene gun and the microinjection methodwhich comprises injecting a gene directly into cells using amicrocapillary or the like. In carrying out the gene expression inducingmethod provided by the present invention, any of such gene transfermethods can be utilized.

In thus-obtained transformant plant, the occurrence of the geneintegrated into a nuclear chromosome and of the gene product can beeasily confirmed by PCR and western analysis, respectively.

While it is possible to obtain each transformant plant by transferring agene for an actinomycete autogenous regulatory factor receptor protein(repressor) or a gene placed under the control of a target sequence(operator) for the receptor protein, it is possible to obtain atransformant plant having both genes transferred therein by the methodutilizing plasmid vectors differing in selective marker generespectively for successive transformation procedures or by the methodutilizing a plasmid vector with both genes incorporated therein.

In accordance with the present invention, the actinomycete autogenousregulatory factor is administered to thus-transformed plant to therebyinduce the expression of a gene placed under the control of the operatorat a site of administration of the actinomycete autogenous regulatoryfactor.

For example, by providing tobacco plants and cultured tobacco cells withcharacters of the repressor BarA (receptor protein for VB) and theoperator BARE-3 (one of the target sequences for BarA) which constitutea gene expression inducing system with the actinomycete Streptomycesvirginiae autogenous regulatory factor VB as an inducer by genetransfer, and administering VB to thus-transformed tobacco plants andcultured tobacco cells, the expression of a gene placed under thecontrol of BARE-3 could be induced at a site of administration of VB.For example, the expression of the gene placed under the control ofBARE-3 could be satisfactorily induced at a VB concentration as low as100 nM.

Owing to their relatively low molecular weights of about 200 in additionto their hydrophobic structures, the actinomycete autogenous regulatoryfactors can easily pass through the cell membrane. Therefore, they arevery suited for use as inducers desired to be rapidly absorbed intoplants.

The actinomycete autogenous regulatory factors have no toxicity toplants. For example, VB shows no toxicity to plants even at aconcentration of 10 μM.

In accordance with the present invention, it is possible to produceuseful transformant plants and efficiently utilize the transformantplants by choosing the gene to be placed under the control of theoperator. For example, by placing a gene capable of providing a plantwith fertility under the control of the operator, it is possible tocontrol the fertility of the above transformant plant by administeringan actinomycete autogenous regulatory factor to that plant. Such a plantcan be utilized, for example, as a host for transformation to therebypreventing transformant plants from otherwise spreading through thenatural environment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of analysis, by the western blotting method, asto whether the BarA protein was accumulated in cultured tobacco cellstransformed in Example 3 by providing them with the characters of therepressor BarA (receptor protein for VB) constituting a gene expressioninducing system with the actinomycete Streptomyces virginiae autogenousregulatory factor VB as an inducer by gene transfer.

(Explanation of Symbols)

M: Molecular weight marker

R:10 ng of the BarA protein produced using a recombinant Escherichiacoli strain and purified 30, 21, 27: Identification numbers of thecultured tobacco cell transformant clones obtained

B: Cultured tobacco cell BY2

T: The transiently transformed cultured tobacco cell protoplast obtained(Example 5)

Arrow: Position of the band indicative of the BarA protein

FIG. 2 shows the results of an examination as to whether the expressionof the GUS reporter gene placed under the control of BARE-3 was inducedin cultured tobacco cells transformed in Example 4 by providing themwith the characters of the repressor BarA (receptor protein for VB) andoperator BARE-3 (one of the target sequences for BarA) constituting agene expression inducing system with the actinomycete Streptomycesvirginiae autogenous regulatory factor VB as the inducer by genetransfer when VB was administered to the cultured tobacco celltransformants.

(Explanation of Symbols)

GUS specific activity (ordinate axis of the graph): used for theevaluation of the GUS gene expression activity (units: [nmol 4MU/min/mgprotein])

30-16, 30-17, 30-23, 30-35, 21-5, 21-21, 21-22, 27-1, 27-9:

Identification numbers of the cultured tobacco cell transformant clonesobtained

OFF (VB−): VB not added

ON (VB+): VB added (final VB-C₆ concentration:1 μM)

FIG. 3 shows the results of a test (results of test 1) of whether theexpression of the GUS reporter gene placed under the control of BARE-3was induced in cultured tobacco cells transformed in Example 5 byproviding them with the characters of the repressor BarA (receptorprotein for VB) and operator BARE-3 (one of the target sequences forBarA) constituting a gene expression inducing system with theactinomycete Streptomyces virginiae autogenous regulatory factor VB asthe inducer by gene transfer when VB was administered to the transientlytransformed cultured tobacco cells.

(Explanation of Symbols)

Induction rate (ordinate axis of the graph): The gene expressioninducing activity due to VB as expressed in terms of ratio of the GUSgene expression activity when VB was added (final VB-C₆ concentration:1μM, ON) to that without addition of VB (OFF) (GUS gene expressionactivity (ON)/GUS gene expression activity (OFF))35S: When the GUS reporter gene not placed under the control of BARE-3was used for transient transformation 35SD: When the plasmidpCaMV35SD-gus was used as the GUS reporter gene placed under the controlof BARE-3 for transient transformation35SUDD: When the plasmid pCaMV35SUDD-gus was used as the GUS reportergene placed under the control of BARE-3 for transient transformationbarA−: When the barA gene was not used for transient transformationbarA+: When the barA gene was used for transient transformation

FIG. 4 shows the results of a test (results of test 2) of whether theexpression of the GUS reporter gene placed under the control of BARE-3was induced in cultured tobacco cells transformed in Example 6 byproviding them with the characters of the repressor BarA (receptorprotein for VB) and operator BARE-3 (one of the target sequences forBarA) constituting a gene expression inducing system with theactinomycete Streptomyces virginiae autogenous regulatory factor VB asthe inducer by gene transfer when VB was administered to the transientlytransformed cultured tobacco cells.

(Explanation of Symbols)

Induction rate (ordinate axis of the graph): The gene expressioninducing activity due to VB as expressed in terms of ratio of GUS geneexpression activity when VB was added (final VB-C₆ concentration:1 μM,ON) to that without addition of VB (OFF) (GUS gene expression activity(ON)/GUS gene expression activity (OFF))35S: When the GUS reporter gene not placed under the control of BARE-3was used for transient transformation 35SU: When the plasmidpCaMV35SU-gus was used as the GUS reporter gene placed under the controlof BARE-3 for transient transformation35SD: When the plasmid pCaMV35SD-gus was used as the GUS reporter geneplaced under the control of BARE-3 for transient transformation35SUD: When the plasmid pCaMV35SUD-gus was used as the GUS reporter geneplaced under the control of BARE-3 for transient transformation35SUDD: When the plasmid pCaMV35SUDD-gus was used as the GUS reportergene placed under the control of BARE-3 for transient transformation

FIG. 5 shows the results of an examination as to whether the expressionof the GUS reporter gene placed under the control of BARE-3 was inducedin cultured tobacco cells transformed in Example 7 by providing themwith the characters of the repressor BarA (receptor protein for VB) andoperator BARE-3 (one of the target sequences for BarA) constituting agene expression inducing system with the actinomycete Streptomycesvirginiae autogenous regulatory factor VB as the inducer by genetransfer when low concentrations of VB were administered to thetransiently transformed cultured tobacco cells.

(Explanation of Symbols)

Induction rate (ordinate axis of the graph): The gene expressioninducing activity due to VB as expressed in terms of ratio of GUS geneexpression activity when VB was added (ON) to that without addition ofVB (OFF) (GUS gene expression activity (ON)/GUS gene expression activity(OFF))35SD: When the plasmid pCaMV35SD-gus was used as the GUS reporter geneplaced under the control of BARE-3 for transient transformation

-   0 nM: VB not added (OFF)-   10 nM: VB added (ON, final VB-C₆ concentration:10 nM)-   100 nM: VB added (ON, final VB-C₆ concentration:100 n)-   1000 nM: VB added (ON, final VB-C₆ concentration:1000 nM=1 μM)

FIG. 6 shows the results of an examination as to whether the expressionof the GUS reporter gene placed under the control of BARE-3 was inducedin a tobacco plant transformed in Example 10 by providing the same withthe characters of the repressor BarA (receptor protein for VB) andoperator BARE-3 (one of the target sequences for BarA) constituting agene expression inducing system with the actinomycete Streptomycesvirginiae autogenous regulatory factor VB as the inducer by genetransfer when VB was administered to the transformant tobacco plant.

(Explanation of Symbols)

OFF (VB−): VB not added

ON (VB+): VB added (final VB-C₆ concentration:1 μM)

BEST MODES FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention in furtherdetail. These examples are, however, by no means limitative of the scopeof the present invention.

EXAMPLE 1

A plasmid vector was constructed for providing a plant with thecharacter of the repressor BarA (receptor protein for VB) constituting agene expression inducing system with the autogenous regulatory factorvirginiae butanolide (VB) of the actinomycete Streptomyces virginiae asthe inducer by gene transfer, namely for transferring the repressor barAgene into the plant for transformation thereof.

For this purpose, the barA gene coding region was cloned, by PCR, fromthe plasmid pET-p26k [Okamoto et al., J. Biol. Chem. (1995), 270,12319–12326] containing the barA gene shown under SEQ ID NO:1.Chemically synthesized oligo-DNAs respectively comprising the nucleotidesequences shown under SEQ ID NO:8 and SEQ ID NO:9 with the restrictionenzyme BamHI and SacI recognition sites introduced therein wererespectively used as 5′- and 3′-primers for PCR. The fragment amplifiedby PCR was treated with the restriction enzymes BamHI and SacI and theninserted into the plasmid vector pBluescriptII SK(−) [GenBank accessionnumber X52330] for cloning between the restriction enzyme BamHIrecognition site and the SacI recognition site within the multicloningregion (plasmid pbarA). By sequencing, it was confirmed that the barAgene coding region had been correctly cloned.

The binary vector pBI121 [Jefferson et al., EMBO J. (1987), 6,3901–3907] having a structure such that the β-glucuronidase (GUS) genecoding region is connected to a site 3′ downstream of the Cauliflowermosaic virus (CaMV) 35S promoter and having the kanamycin resistancegene as a selective marker gene was deprived of the restriction enzymeBamHI-SacI fragment containing the GUS gene coding region by treatmentwith the restriction enzymes BamHI and SacI. The remaining vectorfragment was subjected to ligation with the restriction enzymeBamHI-SacI fragment containing the barA gene coding region as excisedfrom the plasmid pbarA by treatment with the restriction enzymes BamHIand SacI (binary vector pBICaMV35S-barA).

A plasmid vector for transferring the repressor barA gene into a plantfor transient transformation thereof was also constructed. The startingmaterial used was the plasmid NtADHp-GUS [Nagaya et al., J. Biosci.Bioeng. (2000), 89, 231–235] having a structure such that the GUS genecoding region is connected to a site 3′ downstream of the Nicotianatabacum alcohol dehydrogenase (NtADH) promoter. The NtADH promoter ofthis plasmid is known to exhibit very potent promoter activity intobacco.

The plasmid NtADHp-GUS was deprived of the restriction enzyme BamHI-SacIfragment containing the GUS gene coding region by treatment with therestriction enzymes BamHI and SacI. The remaining vector fragment wassubjected to ligation with the restriction enzyme BamHI-SacI fragmentcontaining the barA gene coding region as excised from the plasmid pbarAby treatment with the restriction enzymes BamHI and SacI (plasmidpNtADH-barA).

The plasmid NtADHp-GUS was deprived of the restriction enzyme BamHI-SacIfragment containing the GUS gene coding region by treatment with therestriction enzymes BamHI and SacI. The remaining vector fragment wasrendered blunt-ended and then subjected to ligation (plasmid pNtADHABS).

The Escherichia coli DH5α strain [supE44, ΔlacU169 (Ø80, lacZΔM15),hsdR17, recA1, endA1, gyrA96, thi-1, relA1] was used as the host in arecombinant DNA experiment. As for the procedure, the standard procedure[Molecular Cloning, Maniatis et al., 1982, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.] was followed.

KOD DNA polymerase [Toyobo Co., Ltd.] was used for the PCR and theconditions employed were as described in the relevant manual.

Sequencing was carried out using a sequencer [P.E. Biosystems Japan Co.,Ltd. ABI PRISM 310 Genetic Analyzer].

EXAMPLE 2

A plasmid vector was constructed for providing a plant with thecharacter of the operator BARE-3 (one of the target sequences for thereceptor protein BarA for VB) constituting a gene expression inducingsystem with the actinomycete Streptomyces virginiae autogenousregulatory factor VB as the inducer by gene transfer, namely fortransferring the GUS reporter gene placed under the control of theoperator BARE-3 into the plant for transformation thereof.

Two BARE-3 sequences were disposed in the vicinity of a site 3′downstream and one BARE-3 sequence was disposed in the vicinity of asite 5′ upstream of the TATA box of the CaMV 35S promoter.

For this purpose, a double-stranded DNA fragment comprising thenucleotide sequence shown under SEQ ID NO:7 was synthesized which wasderived from a restriction enzyme EcoRV-XbaI fragment containing theTATA box (5′-TATATAA-3′) of the CaMV 35S promoter (fragment from the762nd nucleotide to 871st nucleotide of the 871 bp CaMV 35S promoter) bysubstitution of BARE-3 (26 bp, shown under SEQ ID NO:3) for each of the26 bp from the 2nd nucleotide to the 27th nucleotide 5′ upstream of theTATA box and the 26 bp from the 2nd nucleotide to the 27th nucleotide 3′downstream of the TATA box and further insertion of BARE-3 (26 bp)between the 27th nucleotide and 28th nucleotide 3′ downstream of theTATA box. Two chemically synthesized oligo-DNAs (100 picomoles each)respectively comprising the nucleotide sequences shown under SEQ IDNO:10 and SEQ ID NO:11 and having 3′ termini complementary to each otherover 16 bp were mixed with 10 μl of a TE solution and the mixture wasmaintained at 95° C. for 3 minutes and then cooled to room temperature.The Escherichia coli DNA polymerase I Klenow fragment was added to 2 μlof the above solution, the total volume was made 40 μl and the resultingreaction mixture was maintained at 37° C. for 30 minutes, the enzyme wasthen inactivated by phenol/chloroform treatment and the ethanolprecipitate from the reaction mixture was dissolved in 5 μl of the TEsolution. A 2-μl portion of this solution was treated with therestriction enzymes EcoRV and XbaI and thus-obtained restriction enzymeEcoRV-XbaI fragment was inserted into the plasmid vector pBluescriptIISK(−) for cloning between the restriction enzyme EcoRV recognition siteand XbaI recognition site within the multicloning region (plasmidpBARE3UDD). Correct positioning of BARE-3 was confirmed by sequencing.

For constructing a CaMV 35S promoter with a structure having two BARE-3sequences disposed in the vicinity of a site 3′ downstream and oneBARE-3 sequence in the vicinity of a site 5′ upstream of the TATA box,the plasmid pBI221 [Jefferson et al., EMBO J (1987), 6, 3901–3907]having a structure such that the GUS gene coding region is connected toa site 3′ downstream of the CaMV 35S promoter was treated with therestriction enzymes HindIII and EcoRV and the thus-excised fragmentcomprising the first nucleotide to 761st nucleotide of the CaMV 35Spromoter was inserted into the plasmid pBARE3UDD between the restrictionenzyme HindIII recognition site and EcoRV recognition site thereof(plasmid pCaMV35SUDD).

The binary vector pBI101HmB [Nakayama et al., Plant Physiol. (2000),122, 1239–1247] having a structure such that the GUS gene coding regionis connected to a site 3′ downstream of the CaMV 35S promoter and alsohaving the hygromycin resistance gene as a selective marker gene wasdeprived of the restriction enzyme HindIII-XbaI fragment containing theCaMV 35S promoter by treatment with the restriction enzymes HindIII andXbaI. The remaining vector fragment was subjected to ligation with therestriction enzyme HindIII-XbaI fragment containing the CaMV 35Spromoter having a structure such that two BARE-3 sequences are disposedin the vicinity of a site 3′ downstream and one BARE-3 sequence isdisposed in the vicinity of a site 5′ upstream of the TATA box asexcised from the plasmid pCaMV35SUDD by treatment with the restrictionenzymes HindIII and XbaI (binary vector pBICaMV35SUDD-gus).

A plasmid vector for transferring the GUS reporter gene placed under thecontrol of the operator BARE-3 into a plant for transient transformationthereof was also constructed.

Two BARE-3 sequences were disposed in the vicinity of a site 3′downstream and one BARE-3 sequence was disposed in the vicinity of asite 5′ upstream of the TATA box of the CaMV 35S promoter.

The plasmid pBI221 was deprived of the restriction enzyme HindIII-XbaIfragment containing the CaMV 35S promoter by treatment with therestriction enzymes HindIII and XbaI. The remaining vector fragment wassubjected to ligation with the restriction enzyme HindIII-XbaI fragmentcontaining the CaMV 35S promoter having a structure such that two BARE-3sequences are disposed in the vicinity of a site 3′ downstream and oneBARE-3 sequence is disposed in the vicinity of a site 5′ upstream of theTATA box as excised from the plasmid pCaMV35SUDD by treatment with therestriction enzymes HindIII and XbaI (plasmid pCaMV35SUDD-gus).

Also constructed in the same manner were plasmid vectors having a CaMV35S promoter with a structure shown under SEQ ID NO:4 and having oneBARE-3 sequence in the vicinity of a site 3′ downstream of the TATA box,a structure shown under SEQ ID NO:5 and having one BARE-3 sequence inthe vicinity of a site 5′ upstream of the TATA box and a structure shownunder SEQ ID NO:6 and having one BARE-3 sequence each in the vicinity ofa site 3′ downstream and in the vicinity of a site 5′ upstream of theTATA box, respectively (plasmids pCaMV35SD-gus, pCaMV35SU-gus andpCaMV35SUD-gus).

EXAMPLE 3

Cultured tobacco cells were provided with the character of the repressorBarA (receptor protein for VB) constituting a gene expression inducingsystem with the actinomycete Streptomyces virginiae autogenousregulatory factor VB as the inducer by gene transfer. In other words,the repressor barA gene was transferred into cultured tobacco cells fortransformation thereof.

For the gene transfer, the Agrobacterium infection method was employed.Agrobacterium was first transformed by transfer of the barA gene andcultured tobacco cells were infected with the transformant Agrobacteriumobtained.

For the gene transfer into Agrobacterium, the electroporation method wasused. Competent cells (50 μl) of the Agrobacterium tumefaciens EHA101strain [Elizanbeth et al., J. Bacteriol. (1986), 168, 1291–1301] wasmixed with 200 ng of the barA gene (Example 1, binary vectorpBICaMV35S-barA) and the mixture was transferred to a cuvette(electrode-to-electrode distance 2 mm) of a gene pulser [Nippon Bio-RadLaboratories]. Pulses were generated between the cuvette electrodesemploying a voltage of 2.5 kV, an electrostatic capacity of 25 μFD and aresistance of 400 Ω. The time constant at the time of pulse generationwas about 10 milliseconds. The whole contents of the pulse-loadedcuvette was spread over a LB medium agar plate containing 100 mg/l ofkanamycin and the plate was allowed to stand in a dark place at 30° C.Two days later, the colonies appearing on the plate were shake-culturedin the dark at 30° C. using 5 ml of LB medium containing 100 mg/l ofkanamycin for 2 days. This culture was used as transformantAgrobacterium culture.

Cultured tobacco BY2 cells (RIKEN Gene Bank Plant Cell Bank RPCNumber 1) [Nagata et al., Methods Enzymol. (1987), 148, 34–39] wereinfected with the transformant Agrobacterium obtained. Cultured tobaccoBY2 cells were subcultured at a dilution rate of about 1/50 and at aboutone-week intervals in the manner of shake culture in the dark at 27° C.using modified LS medium [Nagata et al., Methods Enzymol. (1987), 148,34–39] and cells at the logarithmic growth phase (3 to 5 days after thelast passage) were used for Agrobacterium infection. A 5-ml portion ofthe culture containing tobacco BY2 cells was mixed with 100 μl of thetransformant Agrobacterium culture, the mixture was transferred to adish and this dish was allowed to stand in the dark at 25° C. After 2days, the Agrobacterium was removed from the dish by centrifugation, theremaining tobacco BY2 cells were suspended in 2 to 3 ml of modified LSmedium, the suspension was spread over a modified LS medium-gellan gumplate containing 100 mg/l of kanamycin and 250 mg/l of carbenicillin,and this plate was allowed to stand in the dark at 25° C. Two to threeweeks later, the calli formed on the plate were isolated and subculturedas cultured tobacco cell transformant clones in the presence ofkanamycin and carbenicillin.

Whether the repressor BarA protein had been accumulated in thus-obtainedcultured tobacco cell transformants was analyzed by the western blottingmethod.

Cells of the cultured tobacco cell transformant clones were suspended inan appropriate buffer solution for cell extraction (e.g. 0.1 M KPO4, 2mM EDTA, 5% glycerol, 2 mM DTT, pH 7.8) and disrupted using anultrasonic generator [KK Tomy Seiko's Handy Sonic UR-20P]. The disruptedcell-containing fluid was centrifuged at high-speed and the supernatantobtained was used as the cell extract. The protein concentration (mg/ml)in the cell extract was measured by the Bradford method [Bradford, Anal.Biochem. (1976), 72, 248–254]. An amount of the cell extractcorresponding to 20 μg protein per lane was separated by SDS-PAGE (12.5%polyacrylamide gel), followed by transfer to a PVDF membrane [NipponBio-Rad Laboratories] and reaction with antibodies. Rabbit anti-BarAantibody [Nakano et al., J. Bacteriol. (1998), 180, 3317–3322] was usedas the primary antibody and alkaline phosphatase-labeled goatanti-rabbit IgG antibody as the secondary antibody. Each reaction andwashing procedure was carried out in the presence of 3% skimmed milk.Finally, the membrane was immersed in an alkaline phosphatase reactionmixture (0.017% 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, 1ppm nitro blue tetrazolium, 100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂, pH9.5) for detecting a band developing a color on the membrane. The BarAprotein (10 ng) produced using an Escherichia coli transformant andpurified was used as a control sample.

As a result, the accumulation of the BarA protein was confirmed inseveral cultured tobacco cell transformant clones (FIG. 1).

EXAMPLE 4

Cultured tobacco cells were provided with the characters of therepressor BarA (receptor protein for VB) and operator BARE-3 (one of thetarget sequences for BarA) constituting a gene expression inducingsystem with the actinomycete Streptomyces virginiae autogenousregulatory factor VB as the inducer by gene transfer. In other words,two genes, the repressor barA gene and the GUS reporter gene placedunder the control of the operator BARE-3, were transferred into culturedtobacco cells for transformation thereof.

The GUS reporter gene placed under the control of BARE-3 (Example 2,binary vector pBICaMV35SUDD-gus) was further transferred into two clones(No. 30 and No. 21 shown in FIG. 1) seemingly indicating relatively highlevel acumulation of the BarA protein and one clone (No. 27 shown inFIG. 1) seemingly indicating accumulation of only a small amount of theBarA protein as judged by western analysis among the cultured tobaccocell transformant clones obtained (Example 3) by transferring the barAgene (Example 1, binary vector pBICaMV35S-barA) into cultured tobaccoBY2 cells. Like in Example 3, for the gene transfer, the Agrobacteriuminfection method was employed. Cultured tobacco cell transformant cloneswere selected and subcultured using modified LS medium containing 20mg/l of hygromycin, 100 mg/l of kanamycin and 250 mg/l of carbenicillin.

Whether the expression of the GUS reporter gene placed under the controlof the operator BARE-3 was induced was examined by administering theinducer VB to thus-obtained cultured tobacco cell transformants.

The cultured tobacco cell transformants were subcultured at a dilutionrate of about 1/25 and at about one-week intervals in the manner ofshake culture in the dark at 27° C. using modified LS medium. At thetime of passage, the inducer VB was added, and the GUS gene expressionactivity (evaluated in terms of the GUS activity per unit weight ofprotein, namely the GUS specific activity) of each cell extract preparedfrom cells cultured for 4 days was compared with that of thecorresponding cell extract when VB was not added. For the addition ofVB, a stock solution of VB-C₆ [Nihira, Hakko Kogaku Kaishi (1991), vol.69, 89–105] (10 mg/ml methanol solution) was diluted with water at arate of 1/50 and a 1/1000 volume of the dilution was added to the medium(final VB-C₆ concentration: about 1 μM). Cells were collected from 1 mlof the cell suspension by removing the supernatant by centrifugation andsuspended in 500 μl of a buffer solution for cell extraction (50 mMNaH2PO4/Na2HPO4, 10 mM EDTA, 10 mM 2-mercaptoethanol, pH 7) anddisrupted using an ultrasonic generator [KK Tomy Seiko's Handy SonicUR-20P]. The disrupted cell-containing fluid was centrifuged athigh-speed and the supernatant obtained was used as the cell extract.The protein concentration (mg/ml) in the cell extract was measured bythe Bradford method. The GUS activity [Jefferson et al., EMBO J. (1987),6, 3901–3907] of the cell extract was evaluated based on the amount ofthe fluorescent pigment 4-methylumbelliferone (4MU) formed per unit timeby the enzymatic reaction, at 37° C., of GUS upon addition of 1 mM4-methylumbelliferyl-β-D-glucuronide (4MUG) as the substrate of GUS tothe cell extract. The reaction product 4 MU was quantitated by measuringthe fluorescence at the wavelength 455 nm with excitation at thewavelength 365 nm, and the GUS activity (nmol 4MU/min/ml) was calculatedusing a calibration curve created from standard 4MU. The mean of threeGUS specific activity values [nmol 4MU/min/mg protein] obtained in threeindependent experiments under the same experimental conditions was takenas the GUS gene expression activity under the experimental conditionsmentioned above.

As a result, in a number of the cultured tobacco cell transformantclones, the GUS gene expression activity was higher when VB-C₆ was added(ON (VB+)) than when the same was not added (OFF (VB−)) and thus the GUSgene expression induction by VB could be observed (FIG. 2). Among thecultured tobacco cell transformant clones obtained by furthertransferring the GUS reporter gene placed under the control of BARE-3(Example 2, binary vector pBICaMV35SUDD-gus) into the cultured tobaccocell transformant clones (Example 3, No. 30 and No. 21 shown in FIG. 1)seemingly indicating relatively high level accumulation of the BarAprotein as obtained by transferring the barA gene (Example 1, binaryvector pBICaMV35S-barA) into cultured tobacco BY2 cells, several clones(clone No. 30-derived Nos. 30-16, 30-17, 30-23 and 30-35 and clone No.21-derived Nos. 21-5, 21-21 and 21-22) showed the ratio of the GUS geneexpression activity with addition of VB-C₆ (ON) to that without additionthereof (OFF) (GUS gene expression activity (ON)/GUS gene expressionactivity (OFF)), namely the gene expression inducing activity due to VB(induction rate) of at most about 30 (induction rate≦30). Among thecultured tobacco cell transformant clones obtained by furthertransferring the GUS reporter gene placed under the control of BARE-3(Example 2, binary vector pBICaMV35SUDD-gus) into the cultured tobaccocell transformant clone (Example 3, No. 27 shown in FIG. 1) seeminglyindicating accumulation of only a small amount of the BarA protein asobtained by transferring the barA gene (Example 1, binary vectorpBICaMV35S-barA) into cultured tobacco BY2 cells, some clones (Nos. 27-1and 27-9) showed the gene expression inducing activity due to VB of lessthan 2 (induction rate≦2). Thus, the gene expression inducing activitydue to VB increases as the amount of the BarA protein accumulated in thecultured tobacco cell transformants increased.

In this way, by providing cultured tobacco cells with the characters ofthe repressor BarA (receptor protein for VB) and operator BARE-3 (one ofthe target sequences for BarA) constituting a gene expression inducingsystem with the actinomycete autogenous regulatory factor VB as theinducer by gene transfer and administering VB to thus-obtained culturedtobacco cell transformants, the expression of the gene placed under thecontrol of BARE-3 could be induced at the site of administration of VB.

EXAMPLE 5

Cultured tobacco cells were provided with the characters of therepressor BarA (receptor protein for VB) and operator BARE-3 (one of thetarget sequences for BarA) constituting a gene expression inducingsystem with the actinomycete Streptomyces virginiae autogenousregulatory factor VB as the inducer by gene transfer. In other words,two genes, the repressor barA gene and the GUS reporter gene placedunder the control of the operator BARE-3, were transferred into culturedtobacco cells for transient transformation thereof.

For the gene transfer, the electroporation method was used. Therefore, aprotoplast preparation was prepared from cultured tobacco cells.Cultured tobacco BY2 cells were subcultured at a dilution rate of about1/50 and at about one-week intervals in the manner of shake culture inthe dark at 27° C. using modified LS medium, and cells at thelogarithmic growth phase (3 to 5 days after the last passage) weresuspended in an enzyme solution (0.1% Pectolyase Y23 [KK Yakult], 1%Cellulase “Onozuka” RS [Kikkoman KK], 0.4 M mannitol, pH 5.5). Theenzymatic reaction was allowed to proceed at 30° C. for 2 to 3 hours,during which the cells were dispersed by pipetting at 15-minuteintervals. After confirmation under a microscope of substantiallycomplete dispersion of spherical cells, cells in this state were used asprotoplasts for gene transfer. The protoplasts were washed with 0.4 Mmannitol and then suspended in a buffer solution for electroporation (5mM 2-(N-morpholino)ethanesulfonic acid (MES), 70 mM KCl, 0.3 M mannitol)to a cell density of 3×10⁶/ml. The barA gene (Example 1, plasmidpNtADH-barA; 50 μg), the GUS reporter gene placed under the control ofBARE-3 (Example 2, plasmid pCaMV35SUDD-gus or pCaMV35SD-gus; 5 μg) andthe luciferase (LUC) gene (plasmid pCaMV35S-luc [Millar et al., PlantMol. Biol. Rep. (1992), 10, 324–337]; 1 μg) for monitoring the genetransfer efficiency were mixed up with 500 μl of the protoplastsuspension and the mixture was transferred to a cuvette(electrode-to-electrode distance 4 mm) of a gene pulser [Nippon Bio-RadLaboratories]. Pulses were generated between the cuvette electrodesemploying a voltage of 200 V, an electrostatic capacity of 250 μF and aresistance of 400 Ω. The time constant at the time of pulse generationwas 15 to 20 milliseconds. The protoplasts were swiftly transferred fromthe pulse-loaded cuvette to a dish (diameter 6 cm) and 4.5 ml of amedium (modified LS medium, 10 g/l sucrose, 0.4 M mannitol) was addedthereto.

Whether the expression of the GUS reporter gene placed under the controlof the operator BARE-3 was induced was examined by administering theinducer VB to thus-obtained transiently transformed cultured tobaccocell protoplasts.

The inducer VB was added (final VB-C₆ concentration: 1 μM) to thetransiently transformed cultured tobacco cell protoplast culture in thedish, the dish was allowed to stand in the dark at 25° C. for 20 hoursand then a cell extract was prepared from the protoplasts and the GUSgene expression activity thereof (evaluated in terms of the ratio of theGUS activity to the LUC activity, namely the GUS/LUC value) was comparedwith that found without addition of VB. The addition of VB was carriedout in the same manner as in Example 4. The protoplasts were recoveredfrom the dish, deprived of the supernatant by centrifugation andsuspended in 500 μl of a buffer solution for cell extraction (0.1 MKPO4, 2 mM EDTA, 5% glycerol, 2 mM DTT, pH 7.8) and disrupted using anultrasonic generator [KK Tomy Seiko Handy Sonic UR-20P]. The disruptedcell-containing fluid was centrifuged at high-speed and the supernatantobtained was used as the cell extract. The GUS activity (nmol 4MU/min/ml) of the cell extract was measured in the same manner as inExample 4. The LUC activity of the cell extract was evaluated in termsof the amount of light emitted for 10 seconds as measured using aluminometer [Berthold Institut (Germany) Lumat LB9501] immediately aftermixing of 100 μl of a buffer solution for cell extraction containing 470μM luciferin [Toyo Ink Manufacturing's Pickagene] as the substrate ofLUC with 20 μl of the cell extract at room temperature. The LUC activity(pmol LUC/ml) was calculated using a calibration curve created fromstandard LUC. The mean of three GUS/LUC values (nmol 4MU/min/pmol LUC)obtained in three independent experiments under the same experimentalconditions using the same batch of the protoplast preparation was takenas the GUS gene expression activity under the experimental conditionsmentioned above.

As a result, when the plasmid pCaMV35SUDD-gus or pCaMV35SD-gus was usedas the GUS reporter gene placed under the control of BARE-3 fortransient transformation, the GUS gene expression activity was higherwhen VB-C₆ was added (ON) than when the same was not added (OFF) andthus the GUS gene expression induction by VB could be observed (FIG. 3).The gene expression inducing activities due to VB (induction rate=GUSgene expression activity (ON)/GUS gene expression activity (OFF)) wereinduction rate≈5 (FIG. 3, barA+, 35SUDD) and induction rate≈2 (FIG. 3,barA+, 35SD), respectively. Thus, the gene expression inducing activitydue to VB increased with the increase in the number of BARE-3 sequences.On the other hand, when the barA gene was not used for transienttransformation (the control plasmid pNtADHABS containing no barA genewas used for transient transformation) (FIG. 3, barA−) and when the GUSreporter gene not under the control of BARE-3 (the control plasmidpBI221 containing no BARE-3 was used) was used for transienttransformation (FIG. 3, 35S), no gene expression inducing activity dueto VB was observed in any case (induction rate=1).

As a result of analysis by the same western blotting method as used inExample 3, accumulation of the repressor BarA protein was confirmed inthe transiently transformed cultured tobacco cell protoplasts obtained(FIG. 1, T).

In this way, by providing cultured tobacco cells with the characters ofthe repressor BarA (receptor protein for VB) and operator BARE-3 (one ofthe target sequences for BarA) constituting a gene expression inducingsystem with the actinomycete autogenous regulatory factor VB as theinducer by gene transfer and administering VB to thus-obtainedtransiently transformed cultured tobacco cells, the expression of thegene placed under the control of BARE-3 could be induced at the site ofadministration of VB.

EXAMPLE 6

Cultured tobacco cells were provided with the characters of therepressor BarA (receptor protein for VB) and operator BARE-3 (one of thetarget sequences for BarA) constituting a gene expression inducingsystem with the actinomycete Streptomyces virginiae autogenousregulatory factor VB as the inducer by gene transfer. In other words,the repressor barA gene was transferred into cultured tobacco cells fortransformation thereof and the GUS reporter gene placed under thecontrol of the operator BARE-3 was further transferred intothus-obtained cultured tobacco cell transformant clones for transienttransformation thereof.

The GUS reporter genes placed under the control of BARE-3 (Example 2,plasmid pCaMV35SUDD-gus, pCaMV35SD-gus, pCaMV35SU-gus or pCaMV35SUD-gus)were further transferred into a cultured tobacco cell transformant clone(Example 3, No. 21 shown in FIG. 1) seemingly indicating relatively highlevel accumulation of the BarA protein among the clones obtained bytransferring the barA gene (Example 1, binary vector pBICaMV35S-barA)into cultured tobacco BY2 cells, for transient transformation of thatclone. The transfer of the GUS reporter gene placed under the control ofBARE-3 was carried out in the same manner as in Example 5. Formonitoring the gene transfer efficiency, the LUC gene (plasmidpCaMV35S-luc) was also used for the transient transformation.

Whether the expression of the GUS reporter gene placed under the controlof the operator BARE-3 was induced was examined by administering theinducer VB to thus-obtained transiently transformed cultured tobaccocell protoplasts.

Like in Example 5, the inducer VB was added (final VB-C₆ concentration:1μM) to the transiently transformed cultured tobacco cell protoplastculture in the dish, the dish was allowed to stand in the dark at 25° C.for 20 hours and then a cell extract was prepared from the protoplastsand the GUS gene expression activity thereof (evaluated in terms of theratio of the GUS activity to the LUC activity, namely the GUS/LUC value)was compared with that found without addition of VB.

As a result, when each of the plasmids pCaMV35SUDD-gus, pCaMV35SD-gus,pCaMV35SU-gus and pCaMV35SUS-gus was used as the GUS reporter geneplaced under the control of BARE-3 for transient transformation, the GUSgene expression activity was higher when VB-C₆ was added (ON) than whenthe same was not added (OFF) and thus the GUS gene expression inductionby VB could be observed (FIG. 4). The gene expression inducingactivities due to VB (induction rate=GUS gene expression activity(ON)/GUS gene expression activity (OFF)) were induction rate≈22 (FIG. 4,35SUDD), induction rate≈4 (FIG. 4, 35SD), induction rate≈2 (FIG. 4,35SU) and induction rate≈13 (FIG. 4, 35SUD), respectively. Thus, thegene expression inducing activity due to VB increased with the increasein the number of BARE-3 sequences. Positioning of BARE-3 in the vicinityof a site 3′ downstream of the TATA box resulted in higher geneexpression inducing activity due to VB than positioning in the vicinityof a site 5′ upstream thereof. On the other hand, when the GUS reportergene not under the control of BARE-3 (the control plasmid pBI221containing no BARE-3) was used for transient transformation (FIG. 4,35S), no gene expression inducing activity due to VB was observed(induction rate=1).

In this way, by providing cultured tobacco cells with the characters ofthe repressor BarA (receptor protein for VB) and operator BARE-3 (one ofthe target sequences for BarA) constituting a gene expression inducingsystem with the actinomycete autogenous regulatory factor VB as theinducer by gene transfer and administering VB to thus-obtainedtransiently transformed cultured tobacco cells, the expression of thegene placed under the control of BARE-3 could be induced at the site ofadministration of VB.

EXAMPLE 7

Cultured tobacco cells were provided with the characters of therepressor BarA (receptor protein for VB) and operator BARE-3 (one of thetarget sequences for BarA) constituting a gene expression inducingsystem with the actinomycete Streptomyces virginiae autogenousregulatory factor VB as the inducer by gene transfer and whether theexpression of the GUS reporter gene placed under the control of theoperator BARE-3 was induced was examined by administering the inducer VBin low concentrations to thus-obtained transiently transformed culturedtobacco cells.

Like in Example 6, the GUS reporter gene placed under the control ofBARE-3 (Example 2, plasmid pCaMV35SD-gus) was further transferred into acultured tobacco cell transformant clone (Example 3, No. 21 shown inFIG. 1) seemingly indicating relatively high level accumulation of theBarA protein among the clones obtained by transferring the barA gene(Example 1, binary vector pBICaMV35S-barA) into cultured tobacco BY2cells, for transient transformation of that clone. The inducer VB wasadded to thus-obtained transiently transformed cultured tobacco cellprotoplast culture (final VB-C₆ concentrations:1 μM, 100 nM and 10 nM),cell extracts were prepared from the protoplasts allowed to stand in thedark at 25° C. for 20 hours and the GUS gene expression activitiesthereof (evaluated in terms of the ratio of the GUS activity to the LUCactivity, namely the GUS/LUC value) was compared with that found withoutaddition of VB.

As a result, when the plasmid pCaMV35SD-gus was used as the GUS reportergene placed under the control of BARE-3 for transient transformation,the GUS gene expression activity was higher when VB-C₆ was added (ON) ateach of the concentrations 1 μM, 100 nM and 10 nM than when the same wasnot added (OFF) and thus the GUS gene expression induction by VB couldbe observed (FIG. 5). The gene expression inducing activities due to VB(induction rate=GUS gene expression activity (ON)/GUS gene expressionactivity (OFF)) were induction rate≈5, induction rate≈4 and inductionrate≈1.4, respectively. Thus, the gene expression inducing activity dueto VB could be observed to a satisfactory extent at VB-C₆ concentrationsof not less than 100 nM although it decreased with the decrease in VBconcentration.

In this way, by providing cultured tobacco cells with the characters ofthe repressor BarA (receptor protein for VB) and operator BARE-3 (one ofthe target sequences for BarA) constituting a gene expression inducingsystem with the actinomycete autogenous regulatory factor VB as theinducer by gene transfer and administering VB at a concentration as lowas 100 nM to thus-obtained transiently transformed cultured tobaccocells, the expression of the gene placed under the control of BARE-3could be induced at the site of administration of VB to a satisfactoryextent.

EXAMPLE 8

A tobacco plant was provided with the character of the repressor BarA(receptor protein for VB) constituting a gene expression inducing systemwith the actinomycete Streptomyces virginiae autogenous regulatoryfactor VB as the inducer by gene transfer. In other words, the repressorbarA gene was transferred into a tobacco plant for transformationthereof.

For the gene transfer, the Agrobacterium infection method was employed.Agrobacterium was first transformed by transfer of the barA gene and atobacco plant was infected with the transformant Agrobacterium obtained.

Tobacco (Nicotiana tabacum L.) was infected with the same transformantAgrobacterium as used in Example 3 by the leaf disc method. Square (5 to10 mm) or disk-like leaf sections were cut from several leaves of asterile tobacco plant grown in an MS medium [Murashige et al., Physiol.Plantarum (1962), 15, 473–498] gellan-gum pot and were immersed insterile water in a dish, and several milliliters of the transformantAgrobacterium culture was admixed therewith. The leaf sections weretaken out and arranged face downward on an MS callus medium (containing2 mg/l α-naphthaleneacetic acid and 0.2 mg/l 6-benzyladenine)-gellan gumplate. The leaf sections were recovered from the plate allowed to standin a biotron (25° C., 16 light hours, 8 dark hours) for 2 days, washedwith several portions of sterile water, and arranged face downward on anMS callus medium-gellan gum plate containing 100 mg/l kanamycin and 250mg/l carbenicillin. After 1 to 2 weeks of standing in the biotron, theleaf sections were transferred to and rearranged on an MS shootingmedium (containing 0.02 mg/l α-naphthaleneacetic acid and 1 mg/l6-benzyladenine)-gellan gum plate containing kanamycin andcarbenicillin. Callus formation was confirmed around each leaf section.The plate was allowed to stand in the biotron until shoot was formedfrom the leaf section. The shoots formed were cut off and planted in MSmedium-gellan gum pots containing kanamycin and carbenicillin. Theindividuals that had rooted in the pots allowed to stand in the biotronwere selected, as transformant tobacco plants, in MS medium-gellan gumpots containing 20 mg/l hygromycin, 100 mg/l kanamycin and 250 mg/lcarbenicillin, and maintained by subculture.

EXAMPLE 9

A tobacco plant was provided with the characters of the repressor BarA(receptor protein for VB) and operator BARE-3 (one of the targetsequences for BarA) constituting a gene expression inducing system withthe actinomycete Streptomyces virginiae autogenous regulatory factor VBas the inducer by gene transfer. In other words, the repressor barA genewas transferred into a tobacco plant for transformation thereof and theGUS reporter gene placed under the control of the operator BARE-3 wasfurther transferred into thus-obtained transformant tobacco plant fortransient transformation thereof.

The GUS reporter gene placed under the control of BARE-3 (Example 2,plasmid pCaMV35SD-gus) was further transferred into the transformanttobacco plant (Example 8) obtained by transferring the barA gene(Example 1, binary vector pBICaMV35S-barA) into tobacco (Nicotianatabacum L.), for transient transformation thereof.

For the gene transfer, the electroporation method was used. Therefore,tobacco plant was treated for conversion to protoplasts. Square (5 to 10mm) leaf sections were cut from several leaves of the tobacco plant andsuspended in an enzyme solution (0.1% Pectolyase Y23 [K K Yakult], 1%Cellulase “Onozuka” RS [Kikkoman K K], 0.4 M mannitol, pH 5.5). Theenzymatic reaction was allowed to proceed at room temperature forseveral hours. At the time point when a peeled-off layer was observed onthe leaf surface, the enzyme solution was filtered through a mesh with apore size of 70 μm, and the filtrate was centrifuged. The green mass ofcells settled thereby was used as protoplasts for gene transfer. Theprotoplasts were washed with 0.4 M mannitol and then suspended in abuffer solution for electroporation (5 mM MES, 70 mM KCl, 0.3 Mmannitol) to a cell density of 6×10⁶/ml. The GUS reporter gene placedunder the control of BARE-3 (Example 2, plasmid pCaMV35SD-gus; 10 μg)and the LUC gene (plasmid pCaMV35S-luc; 1 μg) for monitoring the genetransfer efficiency were mixed up with 500 μl of the protoplastsuspension and the mixture was transferred to a cuvette(electrode-to-electrode distance 4 mm) of a gene pulser [Nippon Bio-RadLaboratories]. Pulses were generated between the cuvette electrodesemploying a voltage of 300 V, an electrostatic capacity of 250 μF and aresistance of 400 Ω. The time constant at the time of pulse generationwas about 16 milliseconds. Two equal portions of the protoplasts wereswiftly transferred from the pulse-loaded cuvette to two dishes(diameter 6 cm) and 4.75 ml of a medium (modified LS medium, 10 g/lsucrose, 0.4 M mannitol) was added to each dish.

Whether the expression of the GUS reporter gene placed under the controlof the operator BARE-3 was induced was examined by administering theinducer VB to thus-obtained transiently transformed tobacco protoplasts.

The inducer VB was added (final VB-C₆ concentration: 1 μM) to thetransiently transformed tobacco protoplast culture in one of the dishes,the dish was allowed to stand in the dark at 25° C. for 22 hours andthen a cell extract was prepared from the protoplasts and the GUS geneexpression activity thereof (evaluated in terms of the ratio of the GUSactivity to the LUC activity, namely the GUS/LUC value) was comparedwith that found in the other dish without addition of VB. The additionof VB was carried out in the same manner as in Example 4. Theprotoplasts were recovered from each dish, deprived of the supernatantby centrifugation and suspended in 500 μl of a buffer solution for cellextraction (0.1 M KPO4, 2 mM EDTA, 5% glycerol, 2 mM DTT, pH 7.8) anddisrupted using an ultrasonic generator [KK Tomy Seiko Handy SonicUR-20P]. Each disrupted cell-containing fluid was centrifuged athigh-speed and the supernatant obtained was used as the cell extract.The GUS activity and LUC activity of the cell extract were measured inthe same manner as in Example 4 and Example 5, respectively. Twoindependent experiments were made under the same experimentalconditions.

As a result, when the plasmid pCaMV35SD-gus was used as the GUS reportergene placed under the control of BARE-3 for transient transformation,the GUS gene expression activity was higher when VB-C₆ was added (ON)than when the same was not added (OFF) and thus the GUS gene expressioninduction by VB could be observed. The gene expression inducing activitydue to VB (induction rate=GUS gene expression activity (ON)/GUS geneexpression activity (OFF)) was induction rate≈2 in each experiment.

In this way, by providing a tobacco plant with the characters of therepressor BarA (receptor protein for VB) and operator BARE-3 (one of thetarget sequences for BarA) constituting a gene expression inducingsystem with the actinomycete autogenous regulatory factor VB as theinducer by gene transfer and administering VB to thus-obtainedtransiently transformed tobacco, the expression of the gene placed underthe control of BARE-3 could be induced at the site of administration ofVB.

EXAMPLE 10

A tobacco plant was provided with the characters of the repressor BarA(receptor protein for VB) and operator BARE-3 (one of the targetsequences for BarA) constituting a gene expression inducing system withthe actinomycete Streptomyces virginiae autogenous regulatory factor VBas. the inducer by gene transfer. In other words, two genes, therepressor barA gene and the GUS reporter gene placed under the controlof the operator BARE-3, were transferred into a tobacco plant fortransformation thereof.

The GUS reporter gene placed under the control of BARE-3 (Example 2,binary vector pBICaMV35SUDD-gus) was further transferred into thetransformant tobacco plant (Example 8) obtained by transferring the barAgene (Example 1, binary vector pBICaMV35S-barA) into tobacco (Nicotianatabacum L.). Like in Example 8, for the gene transfer, the Agrobacteriuminfection method was employed. Transformant tobacco plants were selectedon MS medium containing 20 mg/l hygromycin, 100 mg/l kanamycin and 250mg/l carbenicillin and maintained by subculture.

Whether the expression of the GUS reporter gene placed under the controlof the operator BARE-3 was induced was examined by administering theinducer VB to thus-obtained transformant tobacco plant.

Lateral buds of the transformant tobacco plant were subcultured byreplanting in MS medium pots supplemented with VB (final VB-C₆concentration:1 μM) and leaves of the transformant tobacco plant grownin the biotron for about 3 weeks were tested for the GUS gene expressionactivity (evaluated in terms of the degree of staining as observed uponGUS activity staining) for comparison with the activity obtained withoutadding VB. In the GUS activity staining, leaves cut off were immersed ina buffer solution for cell extraction (50 mM NaH2PO4/Na2HPO4, 10 mMEDTA, 10 mM 2-mercaptoethanol, pH 7) containing 1 mM5-bromo-4-chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt(X-gluc) as the substrate of GUS, and the reaction was allowed toproceed overnight at 37° C. The blue pigment formation in the leaves asresulting from the enzymatic reaction of GUS was observed.

As a result, the GUS gene expression activity in the transformanttobacco plant was higher when VB-C₆ was added (ON (VB+)) than when thesame was not added (OFF (VB−)) and thus the GUS gene expressioninduction by VB could be observed (FIG. 6).

In this way, by providing a tobacco plant with the characters of therepressor BarA (receptor protein for VB) and operator BARE-3 (one of thetarget sequences for BarA) constituting a gene expression inducingsystem with the actinomycete autogenous regulatory factor VB as theinducer by gene transfer and administering VB to thus-obtainedtransformant tobacco plant, the expression of the gene placed under thecontrol of BARE-3 could be induced at the site of administration of VB.

INDUSTRIAL APPLICABILITY

The method provided by the invention which comprises providing a plantwith characters of a repressor and operator both constituting a geneexpression inducing system with an actinomycete autogenous regulatoryfactor as an inducer by gene transfer and administering the actinomyceteautogenous regulatory factor to the transformed plant to thereby inducethe expression of a gene placed under the control of the operator at asite of administration of the actinomycete autogenous regulatory factormakes it possible to cause expression of a desired gene at a desiredtime and site, thus enabling even the production, in a plant, of ametabolite otherwise disadvantageous to the growth of the plant. Themethod is also useful in preventing transformant plants from spreadingthrough the environment by controlling the fertility thereof. Thismethod has made it possible to use an inducer excellent incharacteristics and showing gene expression inducing activity in lowerconcentrations as compared with the other known methods of inducing geneexpression in plants and, at the same time, it has opened the way forexpanding the range of alternatives to be used as the inducer.

1. A method of inducing gene expression in a plant which comprisesproviding the plant with a character of a repressor and with a characterof an operator by gene transfer, wherein a gene expression inducingsystem comprises said repressor, said operator, and virginiae butanolideas an inducer, and administering the virginiae butanolide to thetransformed plant to thereby induce, at a site of administration of thevirginiae butanolide, the expression of a gene placed under the controlof the operator, wherein a gene coding for said repressor comprises aregion comprising a nucleotide sequence shown under SEQ ID NO:1, orcomprises a region coding for an amino acid sequence shown under SEQ IDNO:2; a nucleotide sequence of said operator comprises a regioncomprising a nucleotide sequence shown under SEQ ID NO:3; and saidoperator is connected to at least one site 3′ downstream or 5′ upstreamof TATA box of a Cauliflower mosaic virus 35S promoter.
 2. The methodaccording to claim 1, wherein the coding region of the gene coding forsaid repressor is connected to a site 3′ downstream of a plant promoter.3. The method according to claim 2, wherein said plant promoter is aCauliflower mosaic virus 35S promoter.
 4. The method according to claim1, wherein said operator is connected to the TATA box of saidCauliflower mosaic virus 35S promoter, in a manner shown under any ofSEQ ID NO:4 through SEQ ID NO:7.
 5. The method according to claim 1,wherein said gene placed under the control of the operator is a genethat provides the plant with fertility.
 6. A plant transformed by thegene transfer step as recited in claim
 1. 7. Tobacco Nicotiana tabacumL. transformed by the gene transfer step as recited in claim
 1. 8. Acultured plant cell transformed by the gene transfer step as recited inclaim
 1. 9. A cultured tobacco cell transformed by the gene transferstep as recited in claim
 1. 10. A cultured tobacco BY2 cell transformedby the gene transfer step as recited in claim
 1. 11. The methodaccording to claim 1, wherein said gene transfer comprises the step oftransforming the plant with a first vector and with a second vector;wherein said first vector comprises said operator and said gene placedunder the control of said operator, in which said operator is connectedto at least one site 3′ downstream or 5′ upstream of the TATA box of theCauliflower mosaic virus 35S promoter; wherein said second vectorcomprises said gene coding for said repressor.
 12. The method accordingto claim 11, wherein in said second vector, the coding region of saidgene coding for said repressor is connected to a site 3′ downstream of aplant promoter.
 13. The method according to claim 12, wherein said plantpromoter is a Cauliflower mosaic virus 35S promoter.
 14. The methodaccording to claim 11, wherein in said first vector, said operator isconnected to the TATA box of the Cauliflower mosaic virus 35S promoter,in a manner shown under any of SEQ ID NO:4 through SEQ ID NO:7.
 15. Themethod according to claim 11, wherein said gene placed under the controlof the operator is a gene that provides the plant with fertility.